PROJECT SUMMARY Small RNAs (sRNAs) are numerous and carry out intricate regulatory mechanisms, yet their specific roles in bacterial physiology and virulence remain by and large mysterious. Our previous work with two model E. coli sRNAs, SgrS and RyhB, shed light on some of the roles played by sRNAs in bacterial physiology and stress responses and uncovered novel molecular mechanisms of regulation. We showed that a single sRNA can base pair with numerous mRNA targets, and promote the rapid degradation of these mRNAs, with important consequences for key processes including sugar transport and metabolism (SgrS) and iron homeostasis (RyhB). We defined a novel class of competitors, so-called ?sponge? RNAs that bind and modulate the ability of sRNAs to regulate their mRNA targets. In the current proposal, we build on these preliminary studies to address several questions with broad relevance to the sRNA field. 1) What kinetic parameters define sRNA interactions with their targets? Our preliminary data indicate that for SgrS, the target search (where the sRNA finds the appropriate target) is rate limiting and that this parameter is quantitatively different for each distinct sRNA-target interaction. In Aim 1, we will use a novel super-resolution imaging platform that we developed to further interrogate SgrS and RyhB interactions with target mRNAs and define general principles governing sRNA-mRNA interactions. 2) How do sponge RNAs affect the regulatory efficiency of an sRNA on many different targets? Regulation by sRNAs varies from strong effects on some targets to weak effects on other targets. This sets up a regulatory hierarchy that we propose is central to sRNA-mediated control of bacterial stress responses. In Aim 2, we use quantitative parameters defining sRNA regulatory efficiency and determine how these parameters are impacted by the presence and absence of sponge RNAs, which compete with mRNAs for binding to sRNAs. 3) What are the physiological impacts of sponge-mediated regulation of sRNA activity and how prevalent is this level of control? Our preliminary data suggest that sponge RNAs help tune sRNA activity so that sRNAs are only able to act on target mRNAs under the appropriate stress conditions. Though very few have been discovered, we postulate that sponge RNAs are abundant, and that they may be widely used to control many bacterial sRNAs. In Aim 3, we will address both of these issues by examining the physiological consequences of sponge-mediated control of both SgrS and RyhB activity and by conducting genome-wide analyses to identify and characterize novel targets and sponge RNAs for a variety of bacterial sRNAs. The diverse set of techniques that we have developed and optimized will allow us to interrogate sRNA interactions with target mRNAs on a global scale and at the level of single RNA molecules. This will allow us to generate quantitative models for in vivo regulation by sRNAs and elucidate an extensive sRNA regulatory network to produce an sRNA interaction map of greater precision than ever before.